Medical imaging systems

ABSTRACT

A medical imaging system provides simultaneous rendering of visible light and fluorescent images. The system may employ dyes in a small-molecule form that remains in a subject&#39;s blood stream for several minutes, allowing real-time imaging of the subject&#39;s circulatory system superimposed upon a conventional, visible light image of the subject. The system may also employ dyes or other fluorescent substances associated with antibodies, antibody fragments, or ligands that accumulate within a region of diagnostic significance. In one embodiment, the system provides an excitation light source to excite the fluorescent substance and a visible light source for general illumination within the same optical guide that is used to capture images. In another embodiment, the system is configured for use in open surgical procedures by providing an operating area that is closed to ambient light. More broadly, the systems described herein may be used in imaging applications where a visible light image may be usefully supplemented by an image formed from fluorescent emissions from a fluorescent substance that marks areas of functional interest.

GOVERNMENT INTERESTS

The United States Government has certain rights in this inventionpursuant to National Institute of Health Grant # R21CA88245 andDepartment of Energy Grant # DE-FG02-01ER63188.

BACKGROUND OF THE INVENTION

Absorption and fluorescent dyes, such as indocyanine green, have provenuseful for medical imaging applications. Some of the more commonly useddyes share a number of useful characteristics. First, the dyes aresuitable for labeling antibodies or low-molecular-weight ligands ofdiagnostic significance, or otherwise adapted for sequestration orpreferential uptake at a site of interest such as a lesion. The dyes aresafe for injection or other introduction into a live subject. Andfinally, the dyes emit light at a specific wavelength when excited, sothat their location and concentration may be tracked.

A number of imaging systems have been devised to detect and displaythese dyes within living tissue. For example, dyes such as indocyaninegreen have been used to visualize blood flow in eyes. In some cases,such as U.S. Pat. No. 6,293,911 to Imaizumi et al., a dye imaging devicehas been combined with a visible light imaging system. Imaizumidescribes endoscopic tools that generate images of dye-labeledantibodies superimposed over visible light images captured from withinthe body. As a significant disadvantage, the Imaizumi system employs anumber of separate cavities within an endoscopic tool for light sourcesand image capture, thus requiring a greater cross-sectional area for theendoscope. As a further disadvantage, the Imaizumi patent only disclosesendoscopic applications, and may not be suitable for use in opensurgical applications where ambient light may extend into the excitationand/or emission wavelengths of the dye.

There remains a need for improved surgical and diagnostic imaging toolscapable of generating circulatory blood flow images or other functionalimages along with visible light images of a subject.

SUMMARY OF THE INVENTION

A medical imaging system provides simultaneous rendering of visiblelight and fluorescent images. The system may employ dyes in asmall-molecule form that remains in a subject's blood stream for severalminutes, allowing real-time imaging of the subject's circulatory systemsuperimposed upon a conventional, visible light image of the subject.The system may also employ dyes or other fluorescent substancesassociated with antibodies, antibody fragments, or ligands thataccumulate within a region of diagnostic significance. In oneembodiment, the system provides an excitation light source to excite thefluorescent substance and a visible light source for generalillumination within the same optical guide that is used to captureimages. In another embodiment, the system is configured for use in opensurgical procedures by providing an operating area that is closed toambient light. More broadly, the systems described herein may be used inimaging applications where a visible light image may be usefullysupplemented by an image formed from fluorescent emissions from afluorescent substance that marks areas of functional interest.

The medical imaging system may include a visible light source providinglight over a range of wavelengths that includes one or more wavelengthsof visible light, an excitation light source providing light at one ormore wavelengths outside the range of wavelengths of the visible lightsource, the one or more wavelengths selected to excite a fluorescentsubstance, which emits one or more photons at an emission wavelength; anelectronic imaging device; an optical guide having a first end with alens that captures an image of a subject and a second end that couplesthe image to the electronic imaging device; and a filter for couplingthe visible light source and the excitation light source into theoptical guide, the filter reflecting some of the light provided by thevisible light source and some of the light from the excitation lightsource toward the subject, the filter further transmitting some visiblelight from the subject captured by the lens toward the electronicimaging device, and the filter further transmitting the emissionwavelength from the subject captured by the lens toward the electronicimaging device.

In another embodiment, the system may include a visible light sourceilluminating a subject, the visible light source providing a range ofwavelengths including one or more wavelengths of visible light; anexcitation light source illuminating the subject, the excitation lightsource providing an excitation wavelength that is not one of the one ormore wavelengths of visible light; a fluorescent substance introducedinto a circulatory system of the subject, the fluorescent substancebeing soluble in blood carried by the circulatory system and thefluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; an electronic imaging device thatcaptures an image of a field of view that includes some portion of thesubject and the circulatory system of the subject, the image including afirst image obtained from the one or more wavelengths of visible lightand a second image obtained from the emission wavelength; and a displaythat renders the first image and the second image, the second imagebeing displayed at a visible light wavelength.

In another embodiment, the system may include an operating area closedto ambient light, the operating area including a surgical field where asurgical procedure may be performed on a subject; a visible light sourceilluminating the surgical field, the visible light source providing arange of wavelengths including one or more wavelengths of visible light;an excitation light source illuminating the surgical field, theexcitation light source including at least one wavelength outside therange of wavelengths of visible light; a fluorescent substance suitablefor in vivo use, the fluorescent substance fluorescing at an emissionwavelength in response to the at least one wavelength of the excitationlight source, the fluorescent substance being introduced into thesurgical field; an electronic imaging device that captures a visiblelight image of the surgical field and an emission wavelength image ofthe surgical field; and a display that renders the visible light imageand the emission wavelength image of the surgical field, the emissionwavelength image being displayed at a visible light wavelength.

In another embodiment, the system may include a visible light sourcethat illuminates a subject, the visible light source providing a rangeof wavelengths including one or more wavelengths of visible light; anexcitation light source that illuminates the subject at the same timethat the visible light source illuminates the subject, the excitationlight source providing an excitation wavelength that is not one of theone or more wavelengths of visible light; a fluorescent substanceintroduced into a circulatory system of the subject, the fluorescentsubstance being soluble in blood carried by the circulatory system andthe fluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; and an electronic imaging devicethat captures an image of a field of view that includes some portion ofthe subject and the circulatory system of the subject, the imageincluding a first image obtained from the one or more wavelengths ofvisible light and a second image concurrently obtained from the emissionwavelength.

In the embodiments above, the one or more wavelengths of visible lightfrom the visible light source may exclude far-red light, at least one ofthe excitation light source and the emission wavelength including afar-red light wavelength. The filter may be a dichroic mirror placed inthe optical guide at a forty-five degree angle to a central axis of theoptical guide.

The system may include a second filter. The second filter may separatethe emission wavelength from the range of wavelengths from the visiblelight source, the emission wavelength being directed toward a firstoptical transducer of the electronic imaging device and the range ofwavelengths from the visible light source being directed toward a secondoptical transducer of the electronic imaging device. The second filtermay separate the emission wavelength from the range of wavelengths fromthe visible light source, the emission wavelength being directed towarda first optical transducer of the electronic imaging device and therange of wavelengths from the visible light source being directed towarda second optical transducer of the electronic imaging device wherein thesecond optical transducer separately senses at least each one of red,green, and blue light intensities. The second filter may separate theemission wavelength from the range of wavelengths from the visible lightsource, the emission wavelength being directed toward a first opticaltransducer of the electronic imaging device and the range of wavelengthsfrom the visible light source being directed toward a second opticaltransducer of the electronic imaging device wherein the second opticaltransducer separately senses at least each one of cyan, magenta, andyellow light intensities. The second filter may separate the emissionwavelength from the range of wavelengths from the visible light source,the emission wavelength being directed toward a first optical transducerof the electronic imaging device and the range of wavelengths from thevisible light source being directed toward a second optical transducerof the electronic imaging device wherein the second filter includes adichroic mirror that reflects the emission wavelength and transmits theone or more wavelengths of visible light from the visible light source.The second filter may separate the emission wavelength from the range ofwavelengths from the visible light source, the emission wavelength beingdirected toward a first optical transducer of the electronic imagingdevice and the range of wavelengths from the visible light source beingdirected toward a second optical transducer of the electronic imagingdevice wherein the second filter includes a dichroic mirror thatreflects the one or more wavelengths of visible light from the visiblelight source and transmits the emission wavelength. The second filtermay shape the wavelengths of the visible light source.

The electronic imaging device may include at least one charge-coupleddevice. The excitation light source may include a laser. The electronicimaging device may include a video camera sensitive to visible light.the electronic imaging device may include an emission wavelength camera.The electronic imaging device may capture a visible light image and anemission wavelength image, the system further including a processor thatconverts the emission wavelength image to a converted image having oneor more visible light components, and combines the converted image withthe visible light image for display. The electronic imaging device maycapture a visible light image and an emission wavelength image, thesystem further including a processor that converts the emissionwavelength image to a converted image having one or more visible lightcomponents, and superimposes the converted image onto the visible lightimage for display.

The electronic imaging device may capture a visible light image and anemission wavelength image, the visible light image being captured atthirty frames per second and the emission wavelength being captured atfifteen frames per second, the emission wavelength being converted tothirty frames per second for combination with the visible light image.The electronic imaging device may capture a visible light image and anemission wavelength image, wherein the visible light image is capturedat thirty frames per second and the emission wavelength is captured atfifteen frames per second, the visible light image being converted tofifteen frames per second for combination with the emission wavelengthimage.

The system may include a display that displays images captured by theelectronic imaging device. The display may be provided to a physicianfor use during a procedure, the procedure being at least one of adiagnostic procedure or a therapeutic procedure. The display may includea surgical microscope.

The fluorescent substance may label at least one of an antibody, anantibody fragment, or a low-molecular-weight ligand that accumulates ata lesion, the system being used to visualize the lesion. The fluorescentsubstance may be soluble in blood, the system being used to visualize ablood system. The display may render the second image of the circulatorysystem superimposed on the first image of the subject. The fluorescentsubstance may be a fluorescent dye injected into the subject by anintravenous injection. The fluorescent substance may be sprayed onto thesubject. The fluorescent substance may be one or more quantum dots. Thefluorescent substance may include at least one of indocyanine green;fluorescein; methylene blue, and IRDye78-CA.

The fluorescent substance may be a dye having a structure of theformula:

wherein, as valence and stability permit,

-   X represents C(R)₂, S, Se, O, or NR₅;-   R represents H or lower alkyl, or two occurrences of R, taken    together, form a ring together with the carbon atoms through which    they are connected;-   R₁ and R₂ represent, independently, substituted or unsubstituted    lower alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl;-   R₃ represents, independently for each occurrence, one or more    substituents to the ring to which it is attached;-   R₄ represents H, halogen, or a substituted or unsubstituted ether or    thioether of phenol or thiophenol; and-   R₅ represents, independently for each occurrence, substituted or    unsubstituted lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl.

A method as described herein may include illuminating a subject with oneor more wavelengths of visible light; concurrently illuminating thesubject with an excitation wavelength that is not one of the one or morewavelengths of visible light; introducing a fluorescent substance into acirculatory system of the subject, the fluorescent substance beingsoluble in blood carried by the circulatory system and the fluorescentsubstance emitting photons at an emission wavelength in response to theexcitation wavelength; electronically capturing a visible light image ofthe subject; electronically capturing an emission wavelength image ofthe subject that shows the circulatory system; and displayingconcurrently the visible light image of the subject and the emissionwavelength image of the circulatory system.

In another embodiment, a method as described herein may include:enclosing a subject in an operating area closed to ambient light;illuminating the subject with one or more wavelengths of visible light;concurrently illuminating the subject with an excitation wavelength thatis not one of the one or more wavelengths of visible light; introducinga fluorescent substance into the subject, the fluorescent substanceemitting photons at an emission wavelength in response to the excitationwavelength; electronically capturing a visible light image of thesubject; electronically capturing an emission wavelength image of thesubject; and displaying concurrently the visible light image and theemission wavelength.

In another embodiment, a method described herein may include: providingone or more wavelengths of visible light; providing an excitationwavelength that is not one of the one or more wavelengths of visiblelight; introducing a fluorescent substance into a subject, thefluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; providing a laparoscope having afirst optical path that directs the one or more wavelengths of visiblelight toward a subject, a second optical path that directs theexcitation wavelength toward the subject, and a third optical path thatdirects an emission wavelength and the one or more wavelengths ofvisible light from the subject to an imaging device; making an incisionin a body that includes the subject; directing the laparoscope into theincision so that the subject is within a field of view of thelaparoscope; and displaying concurrently a visible light image of thesubject and the emission wavelength image of the subject. At least twoof the first optical path, the second optical path, and the thirdoptical path may be coaxial.

In another embodiment, a method described herein may include: providingone or more wavelengths of visible light; providing an excitationwavelength that is not one of the one or more wavelengths of visiblelight; introducing a fluorescent substance into a subject, thefluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; providing an endoscope having anoptical path for directing images of the subject to an imaging device;coupling the excitation wavelength and the one or more wavelengths ofvisible light into the optical path; directing the endoscope into a bodyso that the subject is within a field of view of the endoscope;capturing an emission wavelength image of the subject and a visiblelight image of the subject at the imaging device; and displayingconcurrently the visible light image of the subject and the emissionwavelength image of the subject.

The fluorescent substance may be a dye having a structure of theformula:

wherein, as valence and stability permit,

-   X represents C(R)₂, S, Se, O, or NR₅;-   R represents H or lower alkyl, or two occurrences of R, taken    together, form a ring together with the carbon atoms through which    they are connected;-   R₁ and R₂ represent, independently, substituted or unsubstituted    lower alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl;-   R₃ represents, independently for each occurrence, one or more    substituents to the ring to which it is attached;-   R₄ represents H, halogen, or a substituted or unsubstituted ether or    thioether of phenol or thiophenol; and-   R₅ represents, independently for each occurrence, substituted or    unsubstituted lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl.

BRIEF DESCRIPTION OF DRAWINGS

The invention will be appreciated more fully from the following furtherdescription thereof, with reference to the accompanying drawings,wherein:

FIG. 1 shows an embodiment of an imaging system for use during opensurgery;

FIG. 2 shows a near-infrared window used by the imaging system;

FIG. 3 shows an embodiment of an imaging system for use in an endoscopictool; and

FIG. 4 shows an image displaying both a circulatory system andsurrounding tissue.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS OF THE INVENTION

To provide an overall understanding of the invention, certainillustrative embodiments will now be described, including a system forgenerating superimposed circulatory and tissue images in video format.However, it will be understood that the methods and systems describedherein can be suitably adapted to other medical imaging applicationswhere visible light tissue images may be usefully displayed withdiagnostic image information obtained from outside the visible lightrange and superimposed onto the visible light image. More generally, themethods and systems described herein may be adapted to any imagingapplication where a visible light image may be usefully displayed with asuperimposed image captured from areas within the visible light imagethat are functionally marked to emit photons outside the visible lightrange by a dye or other material. For example, the systems and methodsare applicable to a wide range of diagnostic or surgical applicationswhere a target pathology, tissue type, or cell may be labeled with afluorescent dye or other fluorescent substance. These and otherapplications of the systems described herein are intended to fall withinthe scope of the invention.

FIG. 1 shows an embodiment of an imaging system for use during opensurgery. The imaging system 100 may include a visible light source 102,and excitation light source 104, a surgical field 106, a dye source 108containing a dye 110, a lens 112, a first filter 114, a second filter116, a third filter 118, a near-infrared camera 120, a video camera 122,an image processing unit 124, and a display 126. In general, the visiblelight source 102 and the excitation light source 104 illuminate thesurgical field 106. The dye 110 may be introduced from the dye source108, such as through injection into the bloodstream of a subject. Animage from the surgical field 106 is then captured by two cameras, thevideo camera 122 capturing a conventional, visible light image of thesurgical field 106 and the near-infrared camera 120 capturing adiagnostic image based upon the distribution of the dye 110 in thesurgical field 106. These images may be combined by the image processingunit 124 and presented on a display 126 where they may be used, forexample, by a surgeon conducting a surgical procedure. Each aspect ofthe system 100 is now described in more detail.

The imaging system 100 may be surrounded by an operating area (notshown) closed to ambient light. As will become clear from the following,many visible light sources such as incandescent lamps, halogen lamps, ordaylight may include a broad spectrum of electromagnetic radiation thatextends beyond the range of visible light detected by the human eye andinto wavelengths used in the present system as a separate opticalchannel for generating diagnostic images. In order to effectively detectemission in these super-visible light wavelengths, it is preferred toenclose the surgical field 106, light sources 102, 104, and cameras 120,122 in an area that is not exposed to broadband light sources. This maybe achieved by using an operating room closed to external light sources,or by using a hood or other enclosure or covering for the surgical field106 that prevents invasion by unwanted spectrum. The visible lightsource 102 may then serve as a light source for the visible light camera122, and also for provide conventional lighting within the visible lightspectrum. As used herein, the term “operating area” is intendedspecifically to refer to an open surgical site that is closed to ambientlight. Endoscopic or laparoscopic applications, as described below, areconfined to surgical procedures within a closed body cavity, and do notinclude an operating area as that term is intended herein.

The visible light source 102 may be, for example, a near-infrareddepleted white light source. This may be a one-hundred fifty Watthalogen lamp with one or more filters to deplete wavelengths greaterthan 700 nanometers (“nm”). Generally, any light source constrained towavelengths between 400 nm and 700 nm may operate as the visible lightsource 102. In certain applications, the excitation light source 104 andresulting emission from the dye 110 may have wavelengths near or below700 μm, as with Cy5 dye, which emits light when excited at 650 nm. Thesenear-red dyes may be used with the present system, however, thisrequires a visible light source 102 that excludes a portion of thevisible light spectrum in which the dye operates, i.e., a far-reddepleted white light source. Similarly, applications using quantum dotsas a fluorescent substance may have absorption or emission wavelengthsanywhere in the visible light spectrum, and a suitable visible lightsource should be depleted at the wavelength(s) of interest. As such, thevisible light source 102 should more generally be understood to be asource of light that includes some, but not necessarily all, of thewavelengths of visible light.

It should also be understood that, in a far-red imaging system orinfrared imaging system such as those noted above, the near-infraredcamera 120 described in the example embodiment will instead be a camerasensitive to the emission wavelength of the dye 110 or other fluorescentsubstance, and that other modifications to light sources, filters andother optics will be appropriate. Similar modifications may be made toisolate a band of wavelengths for dye excitation and emission anywherewithin or outside the visible light range, provided that suitableoptics, cameras, and dyes are available. Other fluorescent substancesmay also be used. For example, quantum dots may emit at visible lightwavelengths, far-red, near-infrared, and infrared wavelengths, and atother wavelengths, typically in response to absorption below theiremission wavelength. Suitable adjustments will be made to the excitationlight source 104 and the emission camera, the near-infrared camera 120in the example embodiment, for such applications. Cameras sensitive tofar-red, near-infrared, and infrared wavelengths are commerciallyavailable.

The excitation light source 104 provides light at a wavelength thatexcites the dye 110. This may be, for example, a laser diode such as a771 nm, 250 mW laser diode system, which may be obtained from LaserComponents of Santa Rosa, Calif. Other single wavelength, narrowband, orbroadband light sources may be used, provided they do not interfere withthe visible light image captured by the video camera 122 or the emissionwavelength of the dye 110. The near-infrared band is generallyunderstood to include wavelengths between 700 nm and 1000 nm, and is auseful wavelength range for a number of readily available excitationlight sources 104 and dyes 110 that may be used with the systemsdescribed herein. Suitable optical coupling and lenses may be providedto direct each of the visible light source 102 and the excitation lightsource 104 at an area of interest within the surgical field 106.

The surgical field 106 may be any area of a subject or patient that isopen for a surgical procedure. This may be, for example, an open chestduring a procedure such as a revascularization or cardiac gene therapy,where visualization of the circulatory system may improve identificationof areas at risk for myocardial infarction. Blood flow visualization maypermit an assessment of coronary arteries during a coronary arterybypass graft, or an assessment of blood flow and viability duringintroduction of genes for endothelial growth factor or fibroblast growthfactor to induce neovascularization within ischemic regions of theheart. More generally, the surgical field 106 may include any areas of apatient's body, such as a region of the body that includes a tumor thatis to be surgically removed, and that is amenable to visualization withfluourescent dyes, such as through the use of labeled antibodies.

The dye source 108 may be any instrument used for injection or otherintroduction of the dye 110 into a subject, such as a hypodermic needleor angiocath. Where, for example, the dye 110 is highly soluble inblood, the dye source 108 may be administered anywhere on the subject,and need not be near the surgical field 106. For example, it has beenfound that IRDye78-CA (the carboxylic acid form of IRDye78), wheninjected intravenously into a live laboratory rat, produced peakvasculature image strength of an open heart approximately 5-10 secondsafter injection, and remained adequate for visualization for over oneminute. In certain embodiments, the dye source 108 may not useinjection. For example, the dye source 108 may spray or otherwise applythe dye 110 to an area of interest. Depending upon the type of dye andthe imaging technique, the dye 110 may be delivered in a discrete dose,or may be continuously or intermittently applied and re-applied by thedye source 108.

The dye 110 may be any dye suitable for use in vivo and havingexcitation and emission wavelengths suitable for other components of thesystem 100. Typically, the dye 110 will be diluted to 25-50 μM forintravenous injection, such as with phosphate buffered saline, which maybe supplemented with Cremophor EL (Sigma) and/or absolute ethanol. Anumber of suitable near-infrared dyes are described below.

‘Acyl’ refers to a group suitable for acylating a nitrogen atom to forman amide or carbamate, a carbon atom to form a ketone, a sulfur atom toform a thioester, or an oxygen atom to form an ester group, e.g., ahydrocarbon attached to a —C(═O)— moiety. Preferred acyl groups includebenzoyl, acetyl, tert-butyl acetyl, pivaloyl, and trifluoroacetyl. Morepreferred acyl groups include acetyl and benzoyl. The most preferredacyl group is acetyl.

The terms ‘amine’ and ‘amino’ are art-recognized and refer to bothunsubstituted and substituted amines as well as ammonium salts, e.g., ascan be represented by the general formula:

wherein R₉, R₁₀, and R′₁₀ each independently represent hydrogen or ahydrocarbon substituent, or R₉ and R₁₀ taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure. In preferred embodiments, none of R₉, R₁₀, andR′₁₀ is acyl, e.g., R₉, R₁₀, and R′₁₀ are selected from hydrogen, alkyl,heteroalkyl, aryl, heteroaryl, carbocyclic aliphatic, and heterocyclicaliphatic. The term ‘alkylamine’ as used herein means an amine group, asdefined above, having at least one substituted or unsubstituted alkylattached thereto. Amino groups that are positively charged (e.g., R′₁₀is present) are referred to as ‘ammonium’ groups. In amino groups otherthan ammonium groups, the amine is preferably basic, e.g., its conjugateacid has a pK_(a) above 7.

The terms ‘amido’ and ‘amide’ are art-recognized as an amino-substitutedcarbonyl, such as a moiety that can be represented by the generalformula:

wherein R₉ and R₁₀ are as defined above. In certain embodiments, theamide will include imides.

‘Alkyl’ refers to a saturated or unsaturated hydrocarbon chain having 1to 18 carbon atoms, preferably 1 to 12, more preferably 1 to 6, morepreferably still 1 to 4 carbon atoms. Alkyl chains may be straight(e.g., n-butyl) or branched (e.g., sec-butyl, isobutyl, or t-butyl).Preferred branched alkyls have one or two branches, preferably onebranch. Preferred alkyls are saturated. Unsaturated alkyls have one ormore double bonds and/or one or more triple bonds. Preferred unsaturatedalkyls have one or two double bonds or one triple bond, more preferablyone double bond. Alkyl chains may be unsubstituted or substituted withfrom 1 to 4 substituents. Preferred alkyls are unsubstituted. Preferredsubstituted alkyls are mono-, di-, or trisubstituted. Preferred alkylsubstituents include halo, haloalkyl, hydroxy, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkyloxycarbonylphenyl, halophenyl), heterocyclyl,and heteroaryl.

The terms ‘alkenyl’ and ‘alkynyl’ refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedabove, but that contain at least one double or triple bond,respectively. When not otherwise indicated, the terms alkenyl andalkynyl preferably refer to lower alkenyl and lower alkynyl groups,respectively. When the term alkyl is present in a list with the termsalkenyl and alkynyl, the term alkyl refers to saturated alkyls exclusiveof alkenyls and alkynyls.

The terms ‘alkoxyl’ and ‘alkoxy’ as used herein refer to an —O-alkylgroup. Representative alkoxyl groups include methoxy, ethoxy, propyloxy,tert-butoxy, and the like. An ‘ether’ is two hydrocarbons covalentlylinked by an oxygen. Accordingly, the substituent of a hydrocarbon thatrenders that hydrocarbon an ether can be an alkoxyl, or another moietysuch as —O-aryl, —O-heteroaryl, —O-heteroalkyl, —O-aralkyl,—O-heteroaralkyl, —O-carbocylic aliphatic, or —O-heterocyclic aliphatic.

The term ‘aralkyl’, as used herein, refers to an alkyl group substitutedwith an aryl group.

‘Aryl ring’ refers to an aromatic hydrocarbon ring system. Aromaticrings are monocyclic or fused bicyclic ring systems, such as phenyl,naphthyl, etc. Monocyclic aromatic rings contain from about 5 to about10 carbon atoms, preferably from 5 to 7 carbon atoms, and mostpreferably from 5 to 6 carbon atoms in the ring. Bicyclic aromatic ringscontain from 8 to 12 carbon atoms, preferably 9 or 10 carbon atoms inthe ring. The term ‘aryl’ also includes bicyclic ring systems whereinonly one of the rings is aromatic, e.g., the other ring is cycloalkyl,cycloalkenyl, or heterocyclyl. Aromatic rings may be unsubstituted orsubstituted with from 1 to about 5 substituents on the ring. Preferredaromatic ring substituents include: halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy, or any combination thereof.More preferred substituents include lower alkyl, cyano, halo, andhaloalkyl.

‘Cycloalkyl ring’ refers to a saturated or unsaturated hydrocarbon ring.Cycloalkyl rings are not aromatic. Cycloalkyl rings are monocyclic, orare fused, spiro, or bridged bicyclic ring systems. Monocycliccycloalkyl rings contain from about 4 to about 10 carbon atoms,preferably from 4 to 7 carbon atoms, and most preferably from 5 to 6carbon atoms in the ring. Bicyclic cycloalkyl rings contain from 8 to 12carbon atoms, preferably from 9 to 10 carbon atoms in the ring.Cycloalkyl rings may be unsubstituted or substituted with from 1 to 4substituents on the ring. Preferred cycloalkyl ring substituents includehalo, cyano, alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Preferred cycloalkyl rings include cyclopentyl, cyclohexyl,cyclohexenyl, cycloheptyl, and cyclooctyl. More preferred cycloalkylrings include cyclohexyl, cycloheptyl, and cyclooctyl.

The term ‘carbonyl’ is art-recognized and includes such moieties as canbe represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R₁₁represents a hydrogen, hydrocarbon substituent, or a pharmaceuticallyacceptable salt, R_(11′) represents a hydrogen or hydrocarbonsubstituent. Where X is an oxygen and R₁₁ or R_(11′) is not hydrogen,the formula represents an ‘ester’. Where X is an oxygen, and R₁₁ is asdefined above, the moiety is referred to herein as a carboxyl group, andparticularly when R₁₁ is a hydrogen, the formula represents a‘carboxylic acid’. Where X is an oxygen, and R_(11′) is hydrogen, theformula represents a ‘formate’. In general, where the oxygen atom of theabove formula is replaced by sulfur, the formula represents a‘thiocarbonyl’ group. Where X is a sulfur and R₁₁ or R_(11′) is nothydrogen, the formula represents a ‘thioester.’ Where X is a sulfur andR₁₁ is hydrogen, the formula represents a ‘thiocarboxylic acid.’ Where Xis a sulfur and R_(11′) is hydrogen, the formula represents a‘thioformate.’ On the other hand, where X is a bond, R₁₁ is nothydrogen, and the carbonyl is bound to a hydrocarbon, the above formularepresents a ‘ketone’ group. Where X is a bond, R₁₁ is hydrogen, and thecarbonyl is bound to a hydrocarbon, the above formula represents an‘aldehyde’ or ‘formyl’ group.

‘Ci alkyl’ is an alkyl chain having i member atoms. For example, C4alkyls contain four carbon member atoms. C4 alkyls containing may besaturated or unsaturated with one or two double bonds (cis or trans) orone triple bond. Preferred C4 alkyls are saturated. Preferredunsaturated C4 alkyl have one double bond. C4 alkyl may be unsubstitutedor substituted with one or two substituents. Preferred substituentsinclude lower alkyl, lower heteroalkyl, cyano, halo, and haloalkyl.

‘Halogen’ refers to fluoro, chloro, bromo, or iodo substituents.Preferred halo are fluoro, chloro and bromo; more preferred are chloroand fluoro.

‘Heteroalkyl’ is a saturated or unsaturated chain of carbon atoms and atleast one heteroatom, wherein no two heteroatoms are adjacent.Heteroalkyl chains contain from 1 to 18 member atoms (carbon andheteroatoms) in the chain, preferably 1 to 12, more preferably 1 to 6,more preferably still 1 to 4. Heteroalkyl chains may be straight orbranched. Preferred branched heteroalkyl have one or two branches,preferably one branch. Preferred heteroalkyl are saturated. Unsaturatedheteroalkyl have one or more double bonds and/or one or more triplebonds. Preferred unsaturated heteroalkyl have one or two double bonds orone triple bond, more preferably one double bond. Heteroalkyl chains maybe unsubstituted or substituted with from 1 to about 4 substituentsunless otherwise specified. Preferred heteroalkyl are unsubstituted.Preferred heteroalkyl substituents include halo, aryl (e.g., phenyl,tolyl, alkoxyphenyl, alkoxycarbonylphenyl, halophenyl), heterocyclyl,heteroaryl. For example, alkyl chains substituted with the followingsubstituents are heteroalkyl: alkoxy (e.g., methoxy, ethoxy, propoxy,butoxy, pentoxy), aryloxy (e.g., phenoxy, chlorophenoxy, tolyloxy,methoxyphenoxy, benzyloxy, alkoxycarbonylphenoxy, acyloxyphenoxy),acyloxy (e.g., propionyloxy, benzoyloxy, acetoxy), carbamoyloxy,carboxy, mercapto, alkylthio, acylthio, arylthio (e.g., phenylthio,chlorophenylthio, alkylphenylthio, alkoxyphenylthio, benzylthio,alkoxycarbonylphenylthio), amino (e.g., amino, mono- and di- C1-C3alkylamino, methylphenylamino, methylbenzylamino, C1-C3 alkylamido,carbamamido, ureido, guanidino).

‘Heteroatom’ refers to a multivalent non-carbon atom, such as a boron,phosphorous, silicon, nitrogen, sulfur, or oxygen atom, preferably anitrogen, sulfur, or oxygen atom. Groups containing more than oneheteroatom may contain different heteroatoms.

‘Heteroaryl ring’ refers to an aromatic ring system containing carbonand from 1 to about 4 heteroatoms in the ring. Heteroaromatic rings aremonocyclic or fused bicyclic ring systems. Monocyclic heteroaromaticrings contain from about 5 to about 10 member atoms (carbon andheteroatoms), preferably from 5 to 7, and most preferably from 5 to 6 inthe ring. Bicyclic heteroaromatic rings contain from 8 to 12 memberatoms, preferably 9 or 10 member atoms in the ring. The term‘heteroaryl’ also includes bicyclic ring systems wherein only one of therings is aromatic, e.g., the other ring is cycloalkyl, cycloalkenyl, orheterocyclyl. Heteroaromatic rings may be unsubstituted or substitutedwith from 1 to about 4 substituents on the ring. Preferredheteroaromatic ring substituents include halo, cyano, lower alkyl,heteroalkyl, haloalkyl, phenyl, phenoxy or any combination thereof.Preferred heteroaromatic rings include thienyl, thiazolyl, oxazolyl,pyrrolyl, purinyl, pyrimidyl, pyridyl, and furanyl. More preferredheteroaromatic rings include thienyl, furanyl, and pyridyl.

‘Heterocyclic aliphatic ring’ is a non-aromatic saturated or unsaturatedring containing carbon and from 1 to about 4 heteroatoms in the ring,wherein no two heteroatoms are adjacent in the ring and preferably nocarbon in the ring attached to a heteroatom also has a hydroxyl, amino,or thiol group attached to it. Heterocyclic aliphatic rings aremonocyclic, or are fused or bridged bicyclic ring systems. Monocyclicheterocyclic aliphatic rings contain from about 4 to about 10 memberatoms (carbon and heteroatoms), preferably from 4 to 7, and mostpreferably from 5 to 6 member atoms in the ring. Bicyclic heterocyclicaliphatic rings contain from 8 to 12 member atoms, preferably 9 or 10member atoms in the ring. Heterocyclic aliphatic rings may beunsubstituted or substituted with from 1 to about 4 substituents on thering. Preferred heterocyclic aliphatic ring substituents include halo,cyano, lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or anycombination thereof. More preferred substituents include halo andhaloalkyl. Heterocyclyl groups include, for example, thiophene,thianthrene, furan, pyran, isobenzofuran, chromene, xanthene,phenoxathin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole,pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole,indole, indazole, purine, quinolizine, isoquinoline, hydantoin,oxazoline, imidazolinetrione, triazolinone, quinoline, phthalazine,naphthyridine, quinoxaline, quinazoline, quinoline, pteridine,carbazole, carboline, phenanthridine, acridine, phenanthroline,phenazine, phenarsazine, phenothiazine, furazan, phenoxazine,pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine,morpholine, lactones, lactams such as azetidinones and pyrrolidinones,sultams, sultones, and the like. Preferred heterocyclic aliphatic ringsinclude piperazyl, morpholinyl, tetrahydrofuranyl, tetrahydropyranyl andpiperidyl. Heterocycles can also be polycycles.

The term ‘hydroxyl’ means —OH.

‘Lower alkyl’ refers to an alkyl chain comprised of 1 to 4, preferably 1to 3 carbon member atoms, more preferably 1 or 2 carbon member atoms.Lower alkyls may be saturated or unsaturated. Preferred lower alkyls aresaturated. Lower alkyls may be unsubstituted or substituted with one orabout two substituents. Preferred substituents on lower alkyl includecyano, halo, trifluoromethyl, amino, and hydroxyl. Throughout theapplication, preferred alkyl groups are lower alkyls. In preferredembodiments, a substituent designated herein as alkyl is a lower alkyl.Likewise, ‘lower alkenyl’ and ‘lower alkynyl’ have similar chainlengths.

‘Lower heteroalkyl’ refers to a heteroalkyl chain comprised of 1 to 4,preferably 1 to 3 member atoms, more preferably 1 to 2 member atoms.Lower heteroalkyl contain one or two non-adjacent heteroatom memberatoms. Preferred lower heteroalkyl contain one heteroatom member atom.Lower heteroalkyl may be saturated or unsaturated. Preferred lowerheteroalkyl are saturated. Lower heteroalkyl may be unsubstituted orsubstituted with one or about two substituents. Preferred substituentson lower heteroalkyl include cyano, halo, trifluoromethyl, and hydroxyl.

‘Mi heteroalkyl’ is a heteroalkyl chain having i member atoms. Forexample, M4 heteroalkyls contain one or two non-adjacent heteroatommember atoms. M4 heteroalkyls containing 1 heteroatom member atom may besaturated or unsaturated with one double bond (cis or trans) or onetriple bond. Preferred M4 heteroalkyl containing 2 heteroatom memberatoms are saturated. Preferred unsaturated M4 heteroalkyl have onedouble bond. M4 heteroalkyl may be unsubstituted or substituted with oneor two substituents. Preferred substituents include lower alkyl, lowerheteroalkyl, cyano, halo, and haloalkyl.

‘Member atom’ refers to a polyvalent atom (e.g., C, O, N, or S atom) ina chain or ring system that constitutes a part of the chain or ring. Forexample, in cresol, six carbon atoms are member atoms of the ring andthe oxygen atom and the carbon atom of the methyl substituent are notmember atoms of the ring.

As used herein, the term ‘nitro’ means —NO₂.

‘Pharmaceutically acceptable salt’ refers to a cationic salt formed atany acidic (e.g., hydroxamic or carboxylic acid) group, or an anionicsalt formed at any basic (e.g., amino or guanidino) group. Such saltsare well known in the art. See e.g., World Patent Publication 87/05297,Johnston et al., published Sep. 11, 1987, incorporated herein byreference. Such salts are made by methods known to one of ordinary skillin the art. It is recognized that the skilled artisan may prefer onesalt over another for improved solubility, stability, formulation ease,price and the like. Determination and optimization of such salts iswithin the purview of the skilled artisan's practice. Preferred cationsinclude the alkali metals (such as sodium and potassium), and alkalineearth metals (such as magnesium and calcium) and organic cations, suchas trimethylammonium, tetrabutylammonium, etc. Preferred anions includehalides (such as chloride), sulfonates, carboxylates, phosphates, andthe like. Clearly contemplated in such salts are addition salts that mayprovide an optical center where once there was none. For example, achiral tartrate salt may be prepared from the compounds of theinvention. This definition includes such chiral salts.

‘Phenyl’ is a six-membered monocyclic aromatic ring that may or may notbe substituted with from 1 to 5 substituents. The substituents may belocated at the ortho, meta or para position on the phenyl ring, or anycombination thereof. Preferred phenyl substituents include: halo, cyano,lower alkyl, heteroalkyl, haloalkyl, phenyl, phenoxy or any combinationthereof. More preferred substituents on the phenyl ring include halo andhaloalkyl. The most preferred substituent is halo.

The terms ‘polycyclyl’ and ‘polycyclic group’ refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, heteroaryls, aryls and/orheterocyclyls) in which two or more member atoms of one ring are memberatoms of a second ring. Rings that are joined through non-adjacent atomsare termed ‘bridged’ rings, and rings that are joined through adjacentatoms are ‘fused rings’.

The term ‘sulfate’ is art-recognized and includes a moiety that can berepresented by the general formula:

in which R₁₀ is as defined above.

A ‘substitution’ or ‘substituent’ on a small organic molecule generallyrefers to a position on a multivalent atom bound to a moiety other thanhydrogen, e.g., a position on a chain or ring exclusive of the memberatoms of the chain or ring. Such moieties include those defined hereinand others as are known in the art, for example, halogen, alkyl,alkenyl, alkynyl, azide, haloalkyl, hydroxyl, carbonyl (such ascarboxyl, alkoxycarbonyl, formyl, ketone, or acyl), thiocarbonyl (suchas thioester, thioacetate, or thioformate), alkoxyl, phosphoryl,phosphonate, phosphinate, amine, amide, amidine, imine, cyano, nitro,azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl,sulfonamido, sulfonyl, silyl, ether, cycloalkyl, heterocyclyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, heteroaralkyl, aralkyl,aryl or heteroaryl. It will be understood by those skilled in the artthat certain substituents, such as aryl, heteroaryl, polycyclyl, alkoxy,alkylamino, alkyl, cycloalkyl, heterocyclyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, and heteroalkynyl, can themselves besubstituted, if appropriate. This invention is not intended to belimited in any manner by the permissible substituents of organiccompounds. It will be understood that ‘substitution’ or ‘substitutedwith’ includes the implicit proviso that such substitution is inaccordance with permitted valence of the substituted atom and thesubstituent, and that the substitution results in a stable compound,e.g., which does not spontaneously undergo transformation such as byrearrangement, cyclization, elimination, hydrolysis, etc.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The abbreviations Me, Et, Ph, Tf, Nf, Ts, and Ms represent methyl,ethyl, phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl, and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations. The abbreviationscontained in said list, and all abbreviations utilized by organicchemists of ordinary skill in the art are hereby incorporated byreference.

For purposes of this invention, the chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 67th Ed., 1986-87, inside cover. Alsofor purposes of this invention, the term ‘hydrocarbon’ is contemplatedto include all permissible compounds or moieties having at least onecarbon-hydrogen bond. In a broad aspect, the permissible hydrocarbonsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic organic compounds which can besubstituted or unsubstituted.

Contemplated equivalents of the compounds described above includecompounds which otherwise correspond thereto, and which have the sameuseful properties thereof, wherein one or more simple variations ofsubstituents are made which do not adversely affect the efficacy of thecompound. In general, the compounds of the present invention may beprepared by the methods illustrated in the general reaction schemes as,for example, described below, or by modifications thereof, using readilyavailable starting materials, reagents and conventional synthesisprocedures. In these reactions, it is also possible to make use ofvariants that are in themselves known, but are not mentioned here.

In certain embodiments, the subject method employs a fluorescent dyehaving a structure of the formula:

wherein, as valence and stability permit,

-   X represents C(R)₂, S, Se, O, or NR₅;-   R represents H or lower alkyl, or two occurrences of R, taken    together, form a ring together with the carbon atoms through which    they are connected;-   R₁ and R₂ represent, independently, substituted or unsubstituted    lower alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl, e.g., optionally substituted by sulfate, phosphate,    sulfonate, phosphonate, halogen, hydroxyl, amino, cyano, nitro,    carboxylic acid, amide, etc., or a pharmaceutically acceptable salt    thereof;-   R₃ represents, independently for each occurrence, one or more    substituents to the ring to which it is attached, such as a fused    ring (e.g., a benzo ring), sulfate, phosphate, sulfonate,    phosphonate, halogen, lower alkyl, hydroxyl, amino, cyano, nitro,    carboxylic acid, amide, etc., or a pharmaceutically acceptable salt    thereof;-   R₄ represents H, halogen, or a substituted or unsubstituted ether or    thioether of phenol or thiophenol; and-   R₅ represents, independently for each occurrence, substituted or    unsubstituted lower alkyl, cycloalkyl, cycloalkylalkyl, aryl, or    aralkyl, e.g., optionally substituted by sulfate, phosphate,    sulfonate, phosphonate, halogen, hydroxyl, amino, cyano, nitro,    carboxylic acid, amide, etc., or a pharmaceutically acceptable salt    thereof.

Dyes representative of this formula include indocyanine green, as wellas:

In certain embodiments wherein two occurrences of R taken together forma ring, the ring is six-membered, e.g., the fluorescent dye has astructure of formula:

wherein X, R₁, R₂, R₃, R₄, and R₅ represent substituents as describedabove.

Dyes representative of this formula include IRDye78, IRDye80, IRDye38,IRDye40, IRDye41, IRDye700, IRDye800, Cy7 (AP Biotech), and compoundsformed by conjugating a second molecule to any such dye, e.g., a proteinor nucleic acid conjugated to IRDye800, IRDye40, or Cy7, etc. The IRDyesare commercially available from Li-Cor Biosciences of Lincoln, Nebr.,and each dye has a specified peak absorption wavelength (also referredto herein as the excitation wavelength) and peak emission wavelengththat may be used to select suitable optical hardware for use therewith.It will be appreciated that other dyes may also be used, including thefar-red dyes noted above, provided suitable adjustments are made to thevisible light imaging components of the system 100, and othernear-infrared dyes or infrared substances such as the previouslymentioned quantum dots. Several specific dyes suited for specificimaging techniques are now described.

IRDye78-CA is useful for imaging the vasculature of the tissues andorgans. The dye in its small molecule form is soluble in blood, and hasan in vivo early half-life of several minutes. This permits multipleinjections during a single procedure. Indocyanine green has similarcharacteristics, but is somewhat less soluble in blood and has a shorterhalf-life. IRDye78 may also be used in other imaging applications, sinceit can be conjugated to tumor-specific ligands for tumor visualization.More generally, IRDye78 may be linked to an antibody, antibody fragment,or ligand associated with a tumor. Presence of the tumor or lesion maythen be visualized using the techniques described above.

As another example, IR-786 partitions efficiently into mitochondriaand/or endoplasmic reticulum in a concentration-dependent manner, thuspermitting blood flow and ischemia visualization in a living heart. Thedye has been successfully applied, for example, to image blood flow inthe heart of a living laboratory rat after a thoracotomy. Moregenerally, IR-786 may be used for non-radioactive imaging of areas ofischemia in the living heart, or other visualization of the viability ofother tissues.

While a number of suitable dyes have been described, it should beappreciated that such fluorescent dyes are examples only, and that moregenerally, any fluorescent substance may be used with the imagingsystems described herein, provided the substance has an emissionwavelength that does not interfere with visible light imaging. Thisincludes the fluorescent dyes described above, as well as substancessuch as quantum dots which may have emission wavelengths above 1000 nm,and may be associated with an antibody, antibody fragment, or ligand andimaged in vivo. All such substances are referred to herein asfluorescent substances, and it will be understood that suitablemodifications may be made to components of the imaging system for usewith any such fluorescent substance.

The lens 112 may be any lens suitable for receiving light from thesurgical field 106 and focusing the light for image capture by thenear-infrared camera 120 and the video camera 122. The lens 112 mayinclude one or more optical coatings suitable for the wavelengths to beimaged, and may provide for manual, electronically-assisted manual, orautomatic control of zoom and focus.

The first filter 114 may be positioned in the image path from the lens112 such that a visible light image having one or more visible lightwavelengths is directed toward the video camera 122, either byreflection or transmittance. An emission image from the excited dye 110passes through the lens 112 and is directed toward the near infraredcamera 120, again either through reflection or transmittance. A numberof arrangements of the cameras 120, 122 and the first filter 114 arepossible, and may involving reflecting or transmitting either thevisible light image or the emission wavelength image.

In one embodiment, IRDye78-CA (carboxylic acid) having a peak absorptionnear 771 nm and a peak emission near 806 nm, is used with the system100. In this embodiment, the first filter 114 may be a 785 nm dichroicmirror that transmits near-infrared light and reflects visible light.The first filter 114 may be positioned within an image path from thelens 112 such that a visible light image of the surgical field 106 isreflected toward the video camera 122 through the third filter 118. Thethird filter 118 may be, for example, a 400 nm-700 nm visible lightfilter. At the same time, the first filter 114 is positioned with theimage path from the lens 112 such that a near-infrared image (i.e., theexcitation wavelength image) is transmitted toward the near-infraredcamera 120 through the second filter 116. The second filter 116 may bean 810 nm+/−20 nm near-infrared emission filter. The filters may bestandard or custom-ordered optical components, which are commerciallyavailable from optical component suppliers. Other arrangements offilters and other optical components may be used with the system 100described herein.

The near-infrared camera 120 may be any still or moving image camerasuitable for capturing images at the emission wavelength of the exciteddye 110. The near-infrared camera may be, for example, an Orca-ERnear-infrared camera with settings of gain 7, 2×2 binning, 640×480 pixelfield of view, and an exposure time of 20 msec and an effective framerate of fifteen frames per second. The Orca-ER is commercially availablefrom Hamamatsu Photonic Systems of Bridgewater, N.J. It will beunderstood that the near-infrared camera 120 of FIG. 1 is only anexample. An infrared camera, a far-red camera, or some other camera orvideo device may be used to capture an emission wavelength image, withthe camera and any associated filters selected according to thewavelength of a corresponding fluorescent substance used with theimaging system. As used herein, the term “emission wavelength camera” isintended to refer to any such camera that may be used with the systemsdescribed herein.

The video camera 122 may be any video camera suitable for capturingimages of the surgical field 106 in the visible light spectrum. In oneembodiment, the video camera 122 is a color video camera model HV-D27,commercially available from Hitachi of Tarrytown, N.Y. The video camera122 may capture red-green-blue (RGB) images at thirty frames per secondat a resolution of 640×480 pixels. More generally, the near-infraredcamera 120 and the video camera 122 may be any device capable ofphotonic detection and conversion to electronic images, including linearphotodiode arrays, charge coupled device arrays, scanningphotomultiplier tubes, and so forth.

The display 126 may be a television, high-definition television,computer monitor, or other display configured to receive and rendersignals from the image processing unit 124. The surgical field 106 mayalso be a neurosurgical site, with a surgical microscope used to viewthe surgical field 106. In this embodiment, the display 126 may be amonocular or binocular eyepiece of the surgical microscope, with thenear-infrared image superimposed on the visible light image in theeyepiece. In another embodiment, the eyepiece may use direct opticalcoupling of the surgical field 106 to the eyepiece for conventionalmicroscopic viewing, with the near-infrared image projected onto theeyepiece using, for example, heads-up display technology.

The image processing unit 124 may include any software and/or hardwaresuitable for receiving images from the cameras 120, 122, processing theimages as desired, and transmitting the images to the display 126. Inone embodiment, the image processing unit 124 is realized in software ona Macintosh computer equipped with a Digi-16 Snapper frame grabber forthe Orca-ER, commercially available from DataCell of North Billerica,Mass., and equipped with a CG-7 frame grabber for the HV-D27,commercially available from Scion of Frederick Md., and using IPLabsoftware, commercially available from Sanalytics of Fairfax, Va. While aMacintosh may be used in one embodiment, any general purpose computermay be programmed to perform the image processing functions describedherein, including an Intel processor-based computer, or a computer usinghardware from Sun Microsystems, Silicon Graphics, or any othermicroprocessor manufacturer.

Generally, the image processing unit 124 should be capable of digitalfiltering, gain adjustment, color balancing, and any other conventionalimage processing functions. The image from the near-infrared camera 120is also typically shifted into the visible light range for display atsome prominent wavelength, e.g., a color distinct from the visible lightcolors of the surgical field 106, so that a superimposed image willclearly depict the dye. The image processing unit 124 may also performimage processing to combine the image from the near-infrared camera 120and the video camera 122. Where the images are displayed side-by-side,this may simply entail rendering the images in suitable locations on acomputer screen. Where the images are superimposed, a frame rateadjustment may be required. That is, if the video camera 122 iscapturing images at the conventional rate of thirty frames per secondand the near-infrared camera 120 is taking still pictures with aneffective frame rate of fifteen frames per second, some additionalprocessing may be required to render the superimposed imagesconcurrently. This may entail either reducing the frame rate of thevideo camera 122 to the frame rate of the near-infrared camera 120either by using every other frame of video data or averaging orotherwise interpolating video data to a slower frame rate. This mayinstead entail increasing the frame rate of the near-infrared imagedata, either by holding each frame of near-infrared data over successiveframes of video data or extrapolating near-infrared data, such as bywarping the near-infrared image according to changes in the video imageor employing other known image processing techniques.

Generally, any combination of software or hardware may be used in theimage processing unit 124. The functions of the image processing unit124 may be realized, for example, in one or more microprocessors,microcontrollers, embedded microcontrollers, programmable digital signalprocessors or other programmable device, along with internal and/orexternal memory such as read-only memory, programmable read-only memory,electronically erasable programmable read-only memory, random accessmemory, dynamic random access memory, double data rate random accessmemory, Rambus direct random access memory, flash memory, or any othervolatile or non-volatile memory for storing program instructions,program data, and program output or other intermediate or final results.The functions may also, or instead, include one or more applicationspecific integrated circuits, programmable gate arrays, programmablearray logic devices, or any other device or devices that may beconfigured to process electronic signals. Any combination of the abovecircuits and components, whether packaged discretely, as a chip, as achipset, or as a die, may be suitably adapted to use with the systemsdescribed herein.

It will further be appreciated that each function of the imageprocessing unit 124 may be realized as computer executable code createdusing a structured programming language such as C, an object-orientedprogramming language such as C++ or Java, or any other high-level orlow-level programming language that may be compiled or interpreted torun on one of the above devices, as well as heterogeneous combinationsof processors, processor architectures, or combinations of differenthardware and software. The image processing unit 124 may be deployedusing software technologies or development environments including a mixof software languages, such as Java, C++, Oracle databases, SQL, and soforth. It will be further appreciated that the functions of the imageprocessing unit 124 may be realized in hardware, software, or somecombination of these.

In one embodiment, the visible light source 102 is a near-infrareddepleted visible light source, the excitation light source 104 is a 771nm, 250 mW laser diode, the dye 110 is indocyanine green or IRDye78-CA,the first filter 114 is a 785 nm dichroic mirror configured to transmitnear-infrared light and reflect visible light, the second filter 116 isan 810 nm+/−20 nm near-infrared emission filter, and the third filter118 is a 400 nm to 700 nm filter. The image processing unit 124 is acomputer with software for image capture from the near-infrared camera120 and the video camera 122, for making suitable color adjustment tothe images from the near-infrared camera 120, for making frame rateadjustments to the video camera 122 image, and for combining the twoimages for superimposed display on the display 126.

The systems described above have numerous surgical applications. Forexample, the system may be deployed as an aid to cardiac surgery, whereit may be used intraoperatively for direct visualization of cardiacblood flow, for direct visualization of myocardium at risk forinfarction, and for image-guided placement of gene therapy and othermedicinals to areas of interest. The system may be deployed as an aid tooncological surgery, where it may be used for direct visualization oftumor cells in a surgical field or for image-guided placement of genetherapy and other medicinals to an area of interest The system may bedeployed as an aid to general surgery for direct visualization of anyfunction amenable to imaging with fluorescent dyes, including blood flowand tissue viability. In dermatology, the system may be used forsensitive detection of malignant cells or other skin conditions, and fornon-surgical diagnosis of dermatological diseases using near-infraredligands and/or antibodies.

FIG. 2 shows a near-infrared window used by the imaging system. Thenear-infrared window 200 is characterized by wavelengths whereabsorbance is at a minimum. The components of living tissue withsignificant near-infrared absorbance include water 204, lipid 208,oxygenated hemoglobin 210, and deoxygenated hemoglobin 212. As shown inFIG. 2, oxygenated hemoglobin 210 and deoxygenated hemoglobin havesignificant absorbance below 700 nm. By contrast, lipids 208 and water204 have significant absorbance above 900 nm. Between 700 nm and 900 nm,these absorbances reach a cumulative minimum referred to as thenear-infrared window 200. While use of excitation and emissionwavelengths outside the near-infrared window 200 is possible, asdescribed in some of the examples above, fluorescence imaging within thenear-infrared window 200 offers several advantages including low tissueautofluorescence, minimized tissue scatter, and relatively deeppenetration depths. While the near-infrared window 200 is one usefulwavelength range for imaging, the systems described herein are notlimited to either excitation or emission wavelengths in this window, andmay employ, for example, far-red light wavelengths below thenear-infrared window 200, or infrared light wavelengths above thenear-infrared window 200, both of which may be captured usingcommercially available imaging equipment.

FIG. 3 shows an embodiment of an imaging system for use in an endoscopictool. The imaging system 300 may include a visible light source 302, andexcitation light source 304, a surgical field 306, a dye source 308containing a dye 310, a lens 312, a first filter 314, a second filter316, a third filter 318, a near-infrared camera 320, a video camera 322,an image processing unit 324, and a display 326. In general, the visiblelight source 302 and the excitation light source 304 illuminate thesurgical field 306. The dye 310 may be introduced from the dye source308, such as through injection into the bloodstream of a subject. Animage from the surgical field 306 is then captured by two cameras, thevideo camera 322 capturing a conventional, visible light image of thesurgical field 306 and the near-infrared camera 320 capturing adiagnostic image based upon the distribution of the dye 310 in thesurgical field 306. These images may be combined by the image processingunit 324 and presented on a display 326 where they may be used, forexample, by a surgeon conducting a surgical procedure. In general, eachof these components may be any of those components similarly describedwith reference to FIG. 1 above. Differences for an endoscopic tool arenow described.

The imaging system 300 for use as an endoscopic tool may further includea first lens/collimator 303 for the visible light source, a secondlens/collimator 305 for the excitation light source 304, an opticalcoupler 307 that combines the excitation light and the visible light, adichroic mirror 309, and an endoscope 311 having a first cavity 313 anda second cavity 315.

The first lens/collimator 303, the second lens/collimator 305, and theoptical coupler 307 serve to combine the excitation light and thevisible light into a single light source. This light source is coupledinto the first cavity 313 through the dichroic mirror 309. In oneembodiment, the dichroic mirror 309 preferably provides fifty percentreflection of light having wavelengths from 400 nm to 700 nm, in orderto optimize an intensity of visible light that reaches the video camera322 after illuminating the surgical field 306 and passing through thedichroic mirror 309 on its return path to the video camera 322. Thedichroic mirror 309 also preferably has greater than ninety percentreflection of wavelength from the excitation light source 304, such asbetween 700 nm and 785 nm, so that these wavelengths are not transmittedto the cameras 320, 322 after reflecting off the surgical field. Usingthis arrangement, visible and excitation light sources 302, 304 sharethe first cavity 313 of the endoscope with the return light path for avisible light image and an emission wavelength image.

The second cavity 315 of the endoscope 311 may be provided for insertionof a tool, such as an optical tool like a laser for irradiation of asite in the surgical field 306, or a physical tool like an instrumentfor taking a biopsy of tissue within the surgical field. By combiningthe optical paths of the imaging system 300 within a single cavity ofthe endoscope 311, the combined gauge of the first cavity 313 forimaging and the second cavity 315 may be advantageously reduced.

The imaging system 300 may instead be used with a laparoscope.Typically, a laparoscope is inserted into a body cavity through anincision, as distinguished from an endoscope which is inserted throughan existing body opening such as the throat or rectum. A laparoscope hasa different form factor than an endoscope, including differentdimensional requirements. Furthermore, use of a laparascope involves atleast one additional step of making an incision into a body so that thelaparascope may be inserted into a body cavity. The laparoscope may beused with any of the imaging systems described above, and the imagingsystem 300 of FIG. 3 in particular would provide the benefit of anarrower bore for illumination and imaging optics.

It will further be appreciated that the imaging system 300 may be usedto simplify imaging devices other than endoscopes and laparoscopes, suchas by providing an integrated, coaxial illumination and image capturedevice using the techniques described above.

In addition to the surgical applications noted above in reference toFIG. 1, the endoscopic tool of FIG. 3 may be used for directvisualization of malignant or pre-malignant areas within a body cavity,or for image-guided placement of gene therapy and other medicinals to anarea of interest within the body cavity.

FIG. 4 shows an image displaying both a circulatory system andsurrounding tissue. As described above, a visible light tissue image 402is captured of tissue within a surgical field. As noted above, thevisible light tissue image 402 may include a subset of visible lightwavelengths when an optical channel for dye imaging includes awavelength within the visible light range. A near-infrared image 404 isalso captured of the same (or an overlapping) field of view of thesurgical field. Although referred to here for convenience as anear-infrared image, it should be clear that the dye-based image 404 mayalso, or instead, employ other wavelengths, such as far-red or infraredwavelengths. The near-infrared image 404 may be shifted to a visiblewavelength for display, preferably using a color that is prominent whensuperimposed on the visible light tissue image 402. The images 402, 404may be frame-rate adjusted as appropriate for video display of thesurgical field.

The images may be displayed separately as the visible light tissue image402 and the near-infrared image 404. Or the images 402, 404 may becombined into a combined image 406 by the image processing unitdescribed above. The combined image 406 may then be used as an aid tothe procedures described above, or to any other surgical or diagnosticprocedure that might benefit from the dye-based imaging techniquesdescribed herein.

It will be appreciated that the above functionality is merelyillustrative, and that other dyes, imaging hardware, and optics may beusefully deployed with the imaging systems described herein. Forexample, an endoscopic tool may employ a still-image imaging system fordiagnostic photography within a body cavity. Or any of the imagingsystems may be used as described above with excitation and/or emissionwavelengths in the far-red spectrum. Through minor adaptations thatwould be clear to one of ordinary skill in the art, the system could beconfigured to image two or more functions (i.e., tumor and blood flow)at the same time that a visible light image is captured by associatingeach function with a different dye having a different emissionwavelength. Non-medical applications exist for the imaging system. Forexample, dyes in a solution form may be sprayed on a mechanicalcomponent to identify oxidation, surface defects, or the like. Dyescould also be used to track gas, steam, or air flow through apressurized system, and in particular to identify leaks around fittingsand valves. These and other arrangements and adaptations of the subjectmatter discussed herein are intended to fall within the scope of theinvention.

Thus, while the invention has been disclosed in connection with thepreferred embodiments shown and described in detail, variousmodifications and improvements thereon will become readily apparent tothose skilled in the art. It should be understood that all mattercontained in the above description or shown in the accompanying drawingsshall be interpreted as illustrative, and not in a limiting sense, andthat the following claims should be interpreted in the broadest senseallowable by law.

1. A system comprising: a visible light source providing light over arange of wavelengths that includes one or more wavelengths of visiblelight; an excitation light source providing light at one or morewavelengths outside the range of wavelengths of the visible lightsource, the one or more wavelengths selected to excite a fluorescentsubstance, which emits one or more photons at an emission wavelength; anelectronic imaging device; an optical guide having a first end with alens that captures an image of a subject and a second end that couplesthe image to the electronic imaging device; and a filter for couplingthe visible light source and the excitation light source into theoptical guide, the filter reflecting some of the light provided by thevisible light source and some of the light from the excitation lightsource toward the subject, the filter further transmitting some visiblelight from the subject captured by the lens toward the electronicimaging device, and the filter further transmitting the emissionwavelength from the subject captured by the lens toward the electronicimaging device.
 2. A system comprising: a visible light sourceilluminating a subject, the visible light source providing a range ofwavelengths including one or more wavelengths of visible light; anexcitation light source illuminating the subject, the excitation lightsource providing an excitation wavelength that is not one of the one ormore wavelengths of visible light; a fluorescent substance introducedinto a circulatory system of the subject, the fluorescent substancebeing soluble in blood carried by the circulatory system and thefluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; an electronic imaging device thatcaptures an image of a field of view that includes some portion of thesubject and the circulatory system of the subject, the image including afirst image obtained from the one or more wavelengths of visible lightand a second image obtained from the emission wavelength; and a displaythat renders the first image and the second image, the second imagebeing displayed at a visible light wavelength.
 3. A system comprising:an operating area closed to ambient light, the operating area includinga surgical field where a surgical procedure may be performed on asubject; a visible light source illuminating the surgical field, thevisible light source providing a range of wavelengths including one ormore wavelengths of visible light; an excitation light sourceilluminating the surgical field, the excitation light source includingat least one wavelength outside the range of wavelengths of visiblelight; a fluorescent substance suitable for in vivo use, the fluorescentsubstance fluorescing at an emission wavelength in response to the atleast one wavelength of the excitation light source, the fluorescentsubstance being introduced into the surgical field; an electronicimaging device that captures a visible light image of the surgical fieldand an emission wavelength image of the surgical field; and a displaythat renders the visible light image and the emission wavelength imageof the surgical field, the emission wavelength image being displayed ata visible light wavelength.
 4. A system comprising: a visible lightsource that illuminates a subject, the visible light source providing arange of wavelengths including one or more wavelengths of visible light;an excitation light source that illuminates the subject at the same timethat the visible light source illuminates the subject, the excitationlight source providing an excitation wavelength that is not one of theone or more wavelengths of visible light; a fluorescent substanceintroduced into a circulatory system of the subject, the fluorescentsubstance being soluble in blood carried by the circulatory system andthe fluorescent substance emitting photons at an emission wavelength inresponse to the excitation wavelength; and an electronic imaging devicethat captures an image of a field of view that includes some portion ofthe subject and the circulatory system of the subject, the imageincluding a first image obtained from the one or more wavelengths ofvisible light and a second image concurrently obtained from the emissionwavelength.
 5. The system of any of claim 1, 2, 3, or 4 wherein the oneor more wavelengths of visible light from the visible light source doesnot include far-red light, at least one of the excitation light sourceand the emission wavelength including a far-red light wavelength.
 6. Thesystem of claim 1 wherein the filter is a dichroic mirror placed in theoptical guide at a forty-five degree angle to a central axis of theoptical guide.
 7. The system of any of claims 1 through 6 furthercomprising a second filter that separates the emission wavelength fromthe range of wavelengths from the visible light source, the emissionwavelength being directed toward a first optical transducer of theelectronic imaging device and the range of wavelengths from the visiblelight source being directed toward a second optical transducer of theelectronic imaging device.
 8. The system of any of claims 1 through 6further comprising a second filter that separates the emissionwavelength from the range of wavelengths from the visible light source,the emission wavelength being directed toward a first optical transducerof the electronic imaging device and the range of wavelengths from thevisible light source being directed toward a second optical transducerof the electronic imaging device wherein the second optical transducerseparately senses at least each one of red, green, and blue lightintensities.
 9. The system of any of claims 1-6 further comprising asecond filter that separates the emission wavelength from the range ofwavelengths from the visible light source, the emission wavelength beingdirected toward a first optical transducer of the electronic imagingdevice and the range of wavelengths from the visible light source beingdirected toward a second optical transducer of the electronic imagingdevice wherein the second optical transducer separately senses at leasteach one of cyan, magenta, and yellow light intensities.
 10. The systemof any of claims 1-6 further comprising a second filter that separatesthe emission wavelength from the range of wavelengths from the visiblelight source, the emission wavelength being directed toward a firstoptical transducer of the electronic imaging device and the range ofwavelengths from the visible light source being directed toward a secondoptical transducer of the electronic imaging device wherein the secondfilter includes a dichroic mirror that reflects the emission wavelengthand transmits the one or more wavelengths of visible light from thevisible light source.
 11. The system of any of claims 1-6 furthercomprising a second filter that separates the emission wavelength fromthe range of wavelengths from the visible light source, the emissionwavelength being directed toward a first optical transducer of theelectronic imaging device and the range of wavelengths from the visiblelight source being directed toward a second optical transducer of theelectronic imaging device wherein the second filter includes a dichroicmirror that reflects the one or more wavelengths of visible light fromthe visible light source and transmits the emission wavelength.
 12. Thesystem of any of claims 1-6 further comprising a second filter thatshapes the wavelengths of the visible light source.
 13. The system ofany of claims 1 through 12 wherein the electronic imaging deviceincludes at least one charge-coupled device.
 14. The system of any ofclaims 1 through 12 wherein the electronic imaging device includes avideo camera sensitive to visible light.
 15. The system of any of claims1 through 12 wherein the electronic imaging device includes an emissionwavelength camera.
 16. The system of any of claims 1 through 12 whereinthe electronic imaging device captures a visible light image and anemission wavelength image, the system further comprising a processorthat converts the emission wavelength image to a converted image havingone or more visible light components, and combines the converted imagewith the visible light image for display.
 17. The system of any ofclaims 1 through 12 wherein the electronic imaging device captures avisible light image and an emission wavelength image, the system furthercomprising a processor that converts the emission wavelength image to aconverted image having one or more visible light components, andsuperimposes the converted image onto the visible light image fordisplay.
 18. The system of any of claims 1 through 12 wherein theelectronic imaging device captures a visible light image and an emissionwavelength image, and wherein the visible light image is captured atthirty frames per second and the emission wavelength is captured atfifteen frames per second, the emission wavelength being converted tothirty frames per second for combination with the visible light image.19. The system of any of claims 1 through 12 wherein the electronicimaging device captures a visible light image and an emission wavelengthimage, and wherein the visible light image is captured at thirty framesper second and the emission wavelength is captured at fifteen frames persecond, the visible light image being converted to fifteen frames persecond for combination with the emission wavelength image.
 20. Thesystem of any of claims 1 through 19 wherein the excitation light sourceincludes a laser.
 21. The system of any of claims 1 through 19 furthercomprising a display that displays images captured by the electronicimaging device.
 22. The system of any of claims 1 through 21 wherein thefluorescent substance labels at least one of an antibody, an antibodyfragment, Or a low-molecular-weight ligand that accumulates at a lesion,the system being used to visualize the lesion.
 23. The system of any ofclaims 1 through 21 wherein the fluorescent substance is soluble inblood, the system being used to visualize a blood system.
 24. The systemof claim 2 wherein the display renders the second image of thecirculatory system superimposed on the first image of the subject. 25.The system of any of claims 1 through 24 wherein the fluorescentsubstance is a fluorescent dye injected into the subject by anintravenous injection.
 26. The system of any of claims 1 through 24wherein the fluorescent substance sprayed onto the subject.
 27. Thesystem of any of claims 1 through 21 wherein the fluorescent substanceis one or more quantum dots.
 28. The system of any of claims 2, 3, or5-21 wherein the display is provided to a physician for use during aprocedure, the procedure being at least one of a diagnostic procedure ora therapeutic procedure.
 29. The system of any of claims 2, 3 or 5-21wherein the display includes a surgical microscope.
 30. The system ofany of claims 1-26 wherein the fluorescent substance includes at leastone of indocyanine green; fluorescein; methylene blue, and IRDye78-CA.31. The system of any of claims 1-21 wherein the fluorescent substanceis a dye having a structure of the formula:

wherein, as valence and stability permit, X represents C(R)₂, S, Se, O,or NR₅; R represents H or lower alkyl, or two occurrences of R, takentogether, form a ring together with the carbon atoms through which theyare connected; R₁ and R₂ represent, independently, substituted orunsubstituted lower alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl,aryl, or aralkyl; R₃ represents, independently for each occurrence, oneor more substituents to the ring to which it is attached; R₄ representsH, halogen, or a substituted or unsubstituted ether or thioether ofphenol or thiophenol; and R₅ represents, independently for eachoccurrence, substituted or unsubstituted lower alkyl, cycloalkyl,cycloalkylalkyl, aryl, or aralkyl.
 32. A system comprising: a visiblelight means for illuminating a subject with one or more wavelengths ofvisible light; an excitation light means for illuminating the subjectwith an excitation wavelength that is not one of the one or morewavelengths of visible light; a fluorescence means introduced into asubject, the fluorescence means for dissolving in blood carried by thecirculatory system and for emitting photons at an emission wavelength inresponse to the excitation wavelength; an imaging means for capturing avisible light image of the subject and an emission wavelength image ofthe circulatory system of the subject; and a display means forconcurrently rendering the visible light image of the subject and theemission wavelength image of the circulatory system.
 33. A methodcomprising: illuminating a subject with one or more wavelengths ofvisible light; concurrently illuminating the subject with an excitationwavelength that is not one of the one or more wavelengths of visiblelight; introducing a fluorescent substance into a circulatory system ofthe subject, the fluorescent substance being soluble in blood carried bythe circulatory system and the fluorescent substance emitting photons atan emission wavelength in response to the excitation wavelength;electronically capturing a visible light image of the subject;electronically capturing an emission wavelength image of the subjectthat shows the circulatory system; and displaying concurrently thevisible light image of the subject and the emission wavelength image ofthe circulatory system.
 34. A method comprising: enclosing a subject inan operating area closed to ambient light; illuminating the subject withone or more wavelengths of visible light; concurrently illuminating thesubject with an excitation wavelength that is not one of the one or morewavelengths of visible light; introducing a fluorescent substance intothe subject, the fluorescent substance emitting photons at an emissionwavelength in response to the excitation wavelength; electronicallycapturing a visible light image of the subject; electronically capturingan emission wavelength image of the subject; and displaying concurrentlythe visible light image and the emission wavelength.
 35. A methodcomprising: providing one or more wavelengths of visible light;providing an excitation wavelength that is not one of the one or morewavelengths of visible light; introducing a fluorescent substance into asubject, the fluorescent substance emitting photons at an emissionwavelength in response to the excitation wavelength; providing alaparoscope having a first optical path that directs the one or morewavelengths of visible light toward a subject, a second optical paththat directs the excitation wavelength toward the subject, and a thirdoptical path that directs an emission wavelength and the one or morewavelengths of visible light from the subject to an imaging device;making an incision in a body that includes the subject; directing thelaparoscope into the incision so that the subject is within a field ofview of the laparoscope; and displaying concurrently a visible lightimage of the subject and the emission wavelength image of the subject.36. The method of claim 35 wherein at least two of the first opticalpath, the second optical path, and the third optical path are coaxial.37. A method comprising: providing one or more wavelengths of visiblelight; providing an excitation wavelength that is not one of the one ormore wavelengths of visible light; introducing a fluorescent substanceinto a subject, the fluorescent substance emitting photons at anemission wavelength in response to the excitation wavelength; providingan endoscope having an optical path for directing images of the subjectto an imaging device; coupling the excitation wavelength and the one ormore wavelengths of visible light into the optical path; directing theendoscope into a body so that the subject is within a field of view ofthe endoscope; capturing an emission wavelength image of the subject anda visible light image of the subject at the imaging device; anddisplaying concurrently the visible light image of the subject and theemission wavelength image of the subject.
 38. The method of any ofclaims 33-37 wherein the fluorescent substance is a dye having astructure of the formula:

wherein, as valence and stability permit, X represents C(R)₂, S, Se, O,or NR₅; R represents H or lower alkyl, or two occurrences of R, takentogether, form a ring together with the carbon atoms through which theyare connected; R₁ and R₂ represent, independently, substituted orunsubstituted lower alkyl, lower alkenyl, cycloalkyl, cycloalkylalkyl,aryl, or aralkyl; R₃ represents, independently for each occurrence, oneor more substituents to the ring to which it is attached; R₄ representsH, halogen, or a substituted or unsubstituted ether or thioether ofphenol or thiophenol; and R₅ represents, independently for eachoccurrence, substituted or unsubstituted lower alkyl, cycloalkyl,cycloalkylalkyl, aryl, or aralkyl.